Retinal Imaging of Prognostic Indicators of Atherosclerosis

描述（由申请人提供）：动脉粥样硬化涉及多种生物分子和细胞介质的参与。当前的成像技术不提供这些参与者的同时成像，因为它们在血管疾病期间协同工作。我们试图利用荧光视网膜成像系统，同时成像多达四个细胞和/或生物分子在视网膜血管系统中的动脉粥样硬化模型的表达。我们的成像剂利用与抗体连接的量子点（QD）的光学特性，在同一成像领域内对不同的生物标志物进行颜色编码。我们假设使用我们的系统可视化视网膜血管中的异常分子表达在生物学和医学中评估动脉粥样硬化具有诊断和预后效用。我们建议将我们的视网膜成像策略与QD相结合，以检测体内视网膜血管系统中的炎症生物标志物，并将表达水平与动脉粥样硬化小鼠模型中近端主动脉中的斑块严重程度相关联。在具体目标1中，我们提出确定视网膜血管系统中动脉粥样硬化成像的候选生物标志物。我们将在3个不同年龄组（6、24和44周）从动脉粥样硬化的ApoE -/-小鼠模型和年龄匹配的对照中切除视网膜，并使用体内注射的QD-抗体缀合物来检测斑块表面上的炎性标志物VCAM-1、MCP-1、MSR和二聚纤维蛋白（D-二聚体），其作为动脉粥样硬化疾病的相对早期和晚期指标。此外，分离的单核细胞和T细胞将使用光谱不同的QD离体标记，并重新输注到小鼠模型和年龄匹配的对照中，并在视网膜组织中定量，以将细胞募集（病变进展的标志物）与分子表达相关联。在相同的动物中，将收获主动脉并探测QD标记的物质，并定量评估免疫细胞浸润和脂质含量。在特定目标2中，我们将在动脉粥样硬化进展过程中对视网膜血管系统进行体内成像。我们将在Aim 1中开发的QD标记的抗体和/或炎性细胞注射到ApoE -/-小鼠模型和年龄匹配的对照中，并在体内对视网膜血管进行成像，以监测分子表达和炎症内皮细胞的细胞募集。我们的实验设计将使我们能够跟踪单个动物随时间的疾病进展。 如果我们的假设是正确的，这种方法具有临床潜力的非侵入性分期动脉粥样硬化的基础上的分子签名。 公共卫生相关性：动脉粥样硬化是一种复杂的疾病，涉及多种细胞类型和蛋白质的不同阶段的起始，进展，并最终斑块破裂，这是负责死亡率和发病率。已经开发了从超声到MRI的许多成像策略，目的是早期检测动脉粥样硬化疾病，使得治疗干预可以在易损斑块破裂之前减缓进展。然而，这些方法不具备检测早期病变所需的分辨率，并且由于结缔组织和脂肪的存在，位于身体深处的动脉（例如主动脉）的成像可能是困难的。此外，目前的成像策略不能检测血管衬里上的斑块中存在的细胞类型和蛋白质的光谱。斑块的“分子标记”如果可检测到，将特别用于动脉粥样硬化疾病的诊断和治疗。例如，病变中巨噬细胞的存在标志着相对“晚期”的斑块更容易破裂，而血管壁上的某些蛋白质，细胞粘附分子，可能发出“早期预警信号”，开始治疗以遏制风险。 在这个提议中，我们寻求使用视网膜成像系统对血管中的动脉粥样硬化进行非侵入性成像。此外，我们将使用光学探针对不同的生物标志物进行颜色编码，例如细胞和细胞粘附分子，使用半导体纳米晶体或量子点具有不同的荧光发射光谱。视网膜为血液循环提供了一个连续可达的非侵入性窗口，可用于在临床中快速安全地获取全身血管的“分子特征”。我们将测量视网膜血管中动脉粥样硬化相关介质的荧光，并将其表达与体内其他难以接近但通常易于形成病变的主要动脉中的表达相关联。然后，将在动脉粥样硬化小鼠模型中对经过验证的生物标志物相关物进行体内成像，以验证我们的成像方法的实用性。

DESCRIPTION (provided by applicant): Atherosclerosis involves the participation of multiple biomolecular and cellular mediators. Current imaging techniques do not provide for the simultaneous imaging of these participants as they work in concert during vascular disease. We seek to utilize a fluorescence retinal imaging system to simultaneously image the expression of up to four cells and/or biomolecules in retinal vasculature in a model of atherosclerosis. Our imaging agents harness the optical properties of quantum dots (QD) linked to antibodies to color-code different biomarkers within the same imaging field. We hypothesize that visualization of abnormal molecular expression in retinal vasculature using our system has diagnostic and prognostic utility in the evaluation of atherosclerosis in biology and medicine. We propose to apply our retinal imaging strategy in conjunction with QD to detect inflammatory biomarkers in retinal vasculature in vivo, and to correlate expression levels with plaque severity in proximal aorta ex vivo in a mouse model of atherosclerosis. In Specific Aim 1 we propose to identify candidate biomarkers for atherosclerotic imaging in retinal vasculature. We will excise retinas from ApoE -/- mouse models of atherosclerosis and age-matched controls at 3 different age groups (6, 24, and 44 weeks) and use QD-antibody conjugates injected in vivo to detect the inflammatory markers VCAM-1, MCP-1, MSR, and dimerized fibrin (D-dimer) on plaque surfaces, which serve as relatively early and late indicators of atherosclerotic disease. In addition, isolated monocytes and T cells will be labeled ex vivo using spectrally-distinct QD and reinfused into mouse models and age-matched controls, and quantified in retinal tissue, to correlate cellular recruitment (a marker of lesion progression) with molecular expression. In the same animals, aortas will be harvested and probed for QD-labeled species and will be quantitatively assessed for immune cell infiltration and lipid content. In Specific Aim 2 we will perform in vivo imaging of retinal vasculature throughout atherosclerotic progression. We will inject QD-labeled antibodies and/or inflammatory cells developed in Aim 1 into ApoE -/- mouse models and age-matched controls and image the retinal vasculature in vivo to monitor molecular expression and cellular recruitment to inflammatory endothelium. Our experimental design will allow us to follow disease progression within a single animal over time. If our hypothesis is correct, this approach has clinical potential for non-invasively staging atherosclerosis based on molecular signatures. PUBLIC HEALTH RELEVANCE: Atherosclerosis is a complex disease involving multiple cell types and proteins in various stages of initiation, progression, and eventually plaque rupture which is responsible for mortality and morbidity. Many imaging strategies ranging from ultrasound to MRI have been developed with the intention of detecting atherosclerotic disease early, such that therapeutic interventions can slow progression before vulnerable plaques rupture. However, these approaches do not possess the resolution necessary to detect early lesions, and imaging of arteries located deep in the body, such as the aorta, can be difficult due to the presence of connective tissue and fat. Furthermore, current imaging strategies are not capable of detecting the spectrum of cell types and proteins present in plaques on vessel linings. The "molecular signature" of plaques, if detectable, would be particularly useful in diagnosis and treatment of atherosclerotic disease. For example, the presence of macrophages in lesions signals a relatively "late stage" plaque which is more likely to rupture, whereas certain proteins on the vessel wall, the cell adhesion molecules, may signal "early warning signs" to start treatment to curb risks. In this proposal, we seek to non-invasively image atherosclerosis in blood vessels using a retinal imaging system. Furthermore, we will use optical probes to color-code different biomarkers, such as cells and cell adhesion molecules, with distinct fluorescent emission spectra using semi-conducting nanocrystals or quantum dots. The retina offers a continuously-accessible, noninvasive window into the circulation, and can be used to rapidly and safely acquire the "molecular signature" of the blood vessels throughout the body in the clinic. We will measure fluorescence due to atherosclerosis-associated mediators in the retinal vessels, and correlate their expression with expression in other inaccessible but major arteries in the body, which are commonly prone to lesion formation. Proven biomarker correlates will then be imaged in vivo in a mouse model of atherosclerosis to validate the utility of our imaging approach.